Malleable demineralized bone composition and method of manufacture

ABSTRACT

A malleable demineralized bone composition consists of cortical bone made from a first portion and a second portion. The first portion of cortical bone is made from cut pieces freeze dried then ground into particles and demineralized then freeze-dried. The second portion of cortical bone is shaved into shavings. The shavings are long thin strips subjected to freeze-drying. The freeze-dried shavings are ground and demineralized and then freeze-dried. A volume of the second portion is placed in a solution of sterile water to create a mixture, the water volume being twice the second portion, the mixture is autoclaved under heat and pressure to form a gelatin, and the first portion is mixed with the gelatin to form a malleable putty or paste.

RELATED APPLICATIONS

This application is a division of co-pending U.S. application Ser. No.15/136,301 entitled, “Malleable Demineralized Bone Composition AndMethod Of Manufacture”, filed Apr. 22, 2016.

TECHNICAL FIELD

This invention is a malleable demineralized bone composition. Morespecifically, a composition that can be formed as a flowable paste or amolded shape retaining structure and a method of manufacture and use ofsaid composition.

BACKGROUND OF THE INVENTION

The manufacture and use of bone allografts from bone tissue is wellknown. The use of particles of various specific sizes and distributionshave been determined to have beneficial characteristics for new bonegrowth in the treatment of osseous defects and bone voids.

The issue of getting the repair composition to stay in position has beenaddressed for various formulations made into malleable paste or putty bythe addition of collagen or other gelatinous materials.

The present invention provides an improvement over those prior artmaterials.

SUMMARY OF THE INVENTION

A malleable demineralized bone composition consists of cortical bonemade from a first portion and a second portion. The first portion ofcortical bone is made from cut pieces freeze dried then ground intoparticles and demineralized then freeze-dried. The second portion ofcortical bone is shaved into shavings. The shavings are long thin stripssubjected to freeze-drying. The freeze-dried shavings are ground anddemineralized and then freeze-dried. A volume of the second portion isplaced in a solution of sterile water to create a mixture, the watervolume being twice the second portion, the mixture is autoclaved underheat and pressure to form a gelatin, and the first portion is mixed withthe gelatin to form a malleable putty or paste.

The second portion is shaved long thin strips cut from cortical boneplates having a length of greater than 5 cm. The cortical shavings areground to a particle size up to 125 microns. The first portion has thecut pieces having a width, a length and a thickness in the range of 1 to4 mm, the pieces are freeze-dried and ground to form the cortical boneground particles of the first portion that are less than 1000 micronsand greater than 100 microns. In a preferred embodiment, the groundparticles of the first portion are sieved to a first size range of 125to 300 microns, a second size range 300 to 500 microns and a third range500 to 850 microns in a ratio of 40:40:20 by weight, respectively.

The malleable demineralized bone composition has a ratio of gelatin fromthe second portion to particles from the first portion of 80:20 byvolume when mixed together. The mixture forms a malleable putty or pastewhich can be packaged in a capped syringe. The packaged mixture isstored at room temperature.

A method of making a malleable demineralized bone composition consistingof cortical bone comprises the steps of: preparing a first portion ofcortical bone by cutting the cortical bone into pieces, freeze-dryingthe pieces and then grinding into particles and demineralizing theground particles and the freeze-drying the demineralized groundparticles to form a first portion of DBM particles; preparing a secondportion of cortical bone by shaving wherein cortical bone plates are cutinto long pieces, cleaned and then shaved to form shavings of corticalbone tissue then freeze-drying the shavings, grinding the freeze-driedshavings to obtain a particle size less than 150 microns anddemineralizing the particles thereafter freeze-drying to form a secondportion of DBM particles; autoclaving a volume of the second portion ofDBM particles mixed with sterile water in a 2:1 ratio by volume for apredetermined time at a pre-set temperature and pressure to form agelatin; cooling the gelatin made from the second portion; and mixingthe freeze-dried ground particles of the first portion with the gelatinfrom the second portion at a ratio of 80:20 or less gelatin to DBM tocreate a malleable putty or paste.

Definitions

Cohesiveness is defined as the capacity of DBM aseptic paste to maintainits shape while immersed in normal saline or water for a minimum of oneminute.

DBM—Demineralized Bone Matrix.

Cryopreserved—Tissue frozen with the addition of, or in a solutioncontaining, a cryoprotectant agent.

Freeze Dried/Lyophilized—Tissue dehydrated for storage by conversion ofthe water content of frozen tissue to a gaseous state under vacuum thatextracts moisture.

Malleability is the ability of DBM aseptic paste to be molded intodifferent shapes with no visible cracks.

Normal Saline—0.9% Sodium Chloride Solution.

PBS—Phosphate Buffered Saline.

SRI—an equipment sterilization company.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described by way of example and with reference tothe accompanying drawings in which:

FIG. 1 shows a photograph of the malleable demineralized bonecomposition rolled into a “ball” like shape in the foreground and twocomposition filled syringes in the background.

FIG. 2 is a photograph of one composition filled syringe packaged in aclear sealed bag.

FIG. 3 is a schematic outline of the overall manufacture process.

FIG. 4 is the subprocess of cutting taken from FIG. 3.

FIG. 5 is the subprocess of grinding and demineralizing taken from FIG.3.

FIG. 6 is the subprocess of gelatin preparation taken from FIG. 3.

FIG. 7 is the subprocess of paste preparation taken from FIG. 3.

FIG. 8 is the subprocess of packaging taken from FIG. 3

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses the manufacturing of a DemineralizedBone Matrix (DBM) aseptic paste or putty derived from human cadavericcortical bone. Cortical bone is obtained from male or female donorswithin suitable age groups. Full body donors with no joint replacementsare preferred. The donors' medical and social history are screened formedical conditions such as osteoporosis and alcohol abuse, which mayhinder the intended purpose of the final product. The demineralizationprocess of bone tissue exposes morphogenetic proteins and otherintrinsic growth factors involved in providing the osteoinductive signalto form new bone. Therefore, the application of DBM aseptic pasteproducts is intended to aid in the treatment of osseous defects and bonevoids. DBM aseptic paste or putty is ready for implantation and does notrequire preoperative preparation such as thawing or mixing. At roomtemperature, DBM aseptic paste or putty is moldable and cohesive.

The DBM paste or putty product is entirely derived from asepticallograft cortical bone. The cortical bone is aseptically cleaned, cut,morselized or shaved, ground, sieved at different sizes, demineralizedand freeze dried to obtain cortical bone particles. Gelatin is obtainedfrom freeze dried ground cortical bone shavings mixed with water. DBMaseptic paste or putty is prepared by mixing ground cortical boneparticles and gelatin. As shown in FIG. 1, final DBM aseptic paste orputty 10 products of 1, 5 or 10 cc are distributed into syringes 20 of 3or 14 ml, packaged in final packaging 40, as shown in FIG. 2, and storedat room temperature until distribution to the end user. To ensure theosteoinductive potential of the final product, every lot ofdemineralized particulate tissue used to prepare DBM aseptic paste istested in vitro using the C2C12 differentiation assay, which has beenshown to have a positive correlation with results obtained in vivo.

FIG. 3 is an outline of the overall manufacturing process. FIGS. 4-8 areeach of the subprocesses.

With reference to FIG. 4, the subprocess of cutting is schematicallyshown. Prior to cutting the donated and approved for processing aseptichuman cadaveric cortical bone, all extraneous material such as musclefibers, adipose tissue, and periosteum are removed from the tissue.Bones are then rinsed a minimum of 3 times with physiological gradenormal saline (0.9% Sodium Chloride). Using a band saw, the bones arecut in a manner that the cortical and cancellous portions are separated.Cortical bone shafts are cut in half longitudinally and placed in basinswith normal saline. Further cleaning and cutting of cortical bone isdivided into 2 subprocesses forming a first and second portion of thecomposition 10, as detailed below.

Cortical bone bulk makes up the first portion 12 of the composition 10.Cortical bone plates are cut into small pieces using a band saw. Thesmall pieces are rinsed a minimum of three times in normal saline andthen placed into a metal container with normal saline. The container iswrapped, placed on a shaker and mechanically agitated for 5 to 10minutes. The bone tissue is then morselized into 1 to 4 mm length andwidth pieces using a morselizer. The tissue is rinsed a minimum of threetimes with normal saline in order to remove any remnants of blood and/orfat deposits. The bone pieces are rinsed with hydrogen peroxide ifrequired for no more than 10 minutes to remove fat/blood. The bonepieces are rinsed a minimum of three times with sterile water to removeany residual hydrogen peroxide. Then, the bone tissue is placed in ametal cube, stored at −80° C. and then freeze dried. The freeze dryingcycle is set to run for 33 hours 50 minutes. It is understood thetiming, ratios and volumes can vary based on the equipment andprocedures used and the above is exemplary of the preferred process forthe inventors' equipment.

Cortical bone shavings makes up the second portion 14 of the composition10. Cortical bone plates are cut into approximately 6.5 cm long pieces.The bone plates are placed in a wash can with normal saline. The washcan is wrapped and agitated for 5 to 10 minutes to remove any blood andadipose tissue. Bone tissues are then rinsed with normal saline as oftenas needed to clean tissue of blood and/or fatty deposits. The bonetissues are shaved using a shaving machine. Cortical bone shavings arecollected in a basin and rinsed with hydrogen peroxide if required forno more than 10 minutes to remove fat/blood if necessary. Cortical boneshavings are rinsed a minimum of three times with sterile water toremove any residual hydrogen peroxide. The shavings are stored at −80°C. and then freeze dried. The freeze drying cycle is set to run for 33hours 50 minutes. It is understood the timing, ratios and volumes canvary based on the equipment and procedures used and the above isexemplary of the preferred process for the inventors' equipment.

As shown in FIG. 5, the next subprocess is grinding-demineralization.

Further processing of cortical bone shavings and cortical bone bulk isdetailed below.

Ground cortical bone shavings; once the freeze drying cycle iscompleted, the cortical shavings are ground and sieved to obtainparticle sizes of 63 to 125 μM. Then, the particulate tissue is mixedwith 0.6 HCL solution at a 20:1 ratio (20 ml of 0.6 HCL to 1 g of bone).The solution containing the tissue is placed on a magnetic stir platefor 19 minutes. After decanting the liquid, the particulate tissue ismixed with sterile water at a 20:1 ratio (20 ml of sterile water to 1 gof bone). The solution containing the tissue is placed on a magneticstir plate for 4 minutes. The process of decanting, mixing andincubating for 4 minutes is repeated with PBS solution. After decantingthe PBS, the particulate tissue is mixed with sterile water at a 20:1ratio (20 ml of sterile water to 1 g of bone). The solution containingthe tissue is placed on a magnetic stir plate for 9 minutes. The waterwaste solution is decanted and the demineralized particulate tissue isstored at −80° C. The demineralized particulate tissue is freeze driedfor 33 hours 50 minutes. At the end of the freeze drying process, asample is collected for Residual Moisture testing.

The grinding-demineralization process of the first portion 12 of thecomposition 10 is similarly, but separately conducted. Cortical bonemix; once the freeze drying cycle is completed, the cortical bulk isground and sieved to obtain particle sizes of 125 to 300 microns, 300 to500 microns and 500 to 850 microns at a ratio of 40:40:20 by weight,respectively. Then, the mix of particulate tissue at different sizes ismixed with 0.7 HCL solution at a 20:1 ratio (20 ml of 0.7 HCL to 1 g ofbone). The solution containing the tissue is placed on a magnetic stirplate for 59 minutes. After decanting the liquid, the particulate tissueis mixed with sterile water at a 20:1 ratio (20 ml of sterile water to 1g of bone). The solution containing the tissue is placed on a magneticstir plate for 4 minutes. The process of decanting, mixing andincubating for 4 minutes is repeated with PBS solution. After decantingthe PBS, the particulate tissue is mixed with sterile water at a 20:1ratio (20 ml of sterile water to 1 g of bone). The solution containingthe tissue is placed on a magnetic stir plate for 9 minutes. The waterwaste solution is decanted and the demineralized particulate tissue (DBMmix) is stored at −80° C. The DBM mix is freeze dried for 33 hours 50minutes. At the end of the freeze drying process, samples are collectedfor residual moisture, residual calcium and osteoinduction testing.

The next subprocess, shown in FIG. 6, is gelatin preparation. Gelatinpreparation and DBM Aseptic Paste Preparation processes are contingentupon positive osteoinduction testing results of the DBM mix. For gelatinpreparation, cortical shavings particles are divided into groups of 100cc, approximately. Each group is placed in a Pyrex glass bottle andmixed with sterile water. The ratio of sterile water to particles is 2:1by volume. This ratio can be varied up or down by adjusting times toaccommodate the altered ratios. In order to prepare gelatin, the mix isautoclaved for 1.25 hours. The autoclaving process includes conditioning(15 minutes), exposure (30 minutes) and drying (30 minutes) steps.Temperature during the exposure step is 121.1° C. and the pressure is30.15 psi. After autoclaving is completed, the gelatin is allowed tocool down to 37° C. in an incubator.

As shown in FIG. 7, DBM aseptic paste preparation is described below.

DBM aseptic paste is prepared by mixing freeze dried DBM mix withgelatin, which must not exceed 40° C. before mixing. The ratio ofgelatin to DBM mix is 80:20 by volume, respectively. The DBM paste isdistributed into 3 ml or 14 ml syringes (1, 5 or 10 cc of product). Thesyringes are capped immediately after the DBM product is put in thesyringe. Surrogate samples of DBM aseptic paste are used for liquidculture sterility testing. Quality Control tests of malleability andcohesiveness are performed using a DBM aseptic paste sample after aminimum of 5 days of preparation. Malleability is the ability of DBMaseptic paste to be molded into different shapes with no visible cracks.Cohesiveness is defined as the capacity of DBM aseptic paste to maintainits shape while immersed in normal saline or water for a minimum of oneminute.

The subprocess shown in FIG. 8 is packaging. Capped syringes containingDBM aseptic paste products are packaged in validated final packaging.The packaged final products are stored at room temperature until theyare distributed to the end user.

Other ranges of bone particle sized and mixture can be employeddepending on the application which, in this example, was boneregeneration. Lower volumes and cell counts may be more suited for lessintrusive bone repairs or more if larger if larger amounts of materialare needed as in a hip defect or repair.

It is understood, the exemplary process can be altered in terms of time,temperature, volume and material ratios and particle size distributionfor a variety of adjustments to the paste or putty consistency. Ideally,the material achieved a low enough viscosity to be pushed through asyringe, but also can be made thick enough to retain its shape whenmolded like putty as the composition 10 in FIG. 1 shaped like a ball.

Variations in the present invention are possible in light of thedescription of it provided herein. While certain representativeembodiments and details have been shown for the purpose of illustratingthe subject invention, it will be apparent to those skilled in this artthat various changes and modifications can be made therein withoutdeparting from the scope of the subject invention. It is, therefore, tobe understood that changes can be made in the particular embodimentsdescribed, which will be within the full intended scope of the inventionas defined by the following appended claims.

What is claimed is:
 1. A method of making a malleable demineralized bonecomposition consisting of demineralized bone matrix (DBM) particles ofcortical bone and water comprises the steps of: preparing a firstportion of cortical bone by cutting the cortical bone into pieces,freeze-drying the pieces and then grinding into particles anddemineralizing the ground particles and thereafter freeze-drying thedemineralized ground particles to form a first portion of DBM particles;preparing a second portion of cortical bone by shaving wherein corticalbone is cut into long pieces, cleaned and then shaved to form shavingsof cortical bone tissue then freeze-drying the shavings, grinding thefreeze-dried shavings to obtain a particle size less than 150 μm anddemineralizing the particles thereafter freeze-drying to form a secondportion of DBM particles; autoclaving a volume of the second portion ofDBM particles mixed with sterile water in a 2:1 ratio by volume for apredetermined time of 1.25 hours including condition of 15 minutes,exposure of 30 minutes and drying of 30 minutes at a pre-set temperatureand pressure during exposure of 121.1 degrees C. and 30.15 psirespectively to form a gelatin; cooling the gelatin made from the secondportion; and mixing the freeze-dried ground particles of the firstportion with the gelatin from the second portion at a ratio of 80:20 orless gelatin to DBM to create a malleable putty or paste.
 2. The methodof making a malleable demineralized bone composition of claim 1 whereinthe second portion is long strips cut from cortical bone, the stripshaving a length of greater than 5 cm.
 3. The method of making amalleable demineralized bone composition of claim 2 wherein the corticalshavings are ground to a particle size up to 125 microns.
 4. The methodof making a malleable demineralized bone composition of claim 1 whereinthe first portion has the cut pieces having a width, a length and athickness in the range of 1 to 4 mm.
 5. The method of making a malleabledemineralized bone composition of claim 1 wherein the cortical boneground particles of the first portion are less than 1000 microns.
 6. Themethod of making a malleable demineralized bone composition of claim 5wherein the ground particles of the first portion greater than 100microns.
 7. The method of making a malleable demineralized bonecomposition of claim 5 wherein the ground particles of the first portionare sieved to a first size range of 125 to 300 microns, a second sizerange 300 to 500 microns and a third range 500 to 850 microns.
 8. Themethod of making a malleable demineralized bone composition of claim 7wherein the size first through third ranges have a ratio of 40:40:20 byweight, respectively.
 9. The method of making a malleable demineralizedbone composition of claim 1 wherein the ratio of gelatin to particlesfrom the first portion is 80:20 by volume.
 10. The method of making amalleable demineralized bone composition of claim 1 wherein the mixtureformed as a malleable putty or paste is packaged in a capped syringe.11. The method of making a malleable demineralized bone composition ofclaim 10 wherein the packaged mixture is stored at room temperature.